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Cell Dec 2008Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the...
Bone marrow hematopoietic stem cells (HSCs) are crucial to maintain lifelong production of all blood cells. Although HSCs divide infrequently, it is thought that the entire HSC pool turns over every few weeks, suggesting that HSCs regularly enter and exit cell cycle. Here, we combine flow cytometry with label-retaining assays (BrdU and histone H2B-GFP) to identify a population of dormant mouse HSCs (d-HSCs) within the lin(-)Sca1+cKit+CD150+CD48(-)CD34(-) population. Computational modeling suggests that d-HSCs divide about every 145 days, or five times per lifetime. d-HSCs harbor the vast majority of multilineage long-term self-renewal activity. While they form a silent reservoir of the most potent HSCs during homeostasis, they are efficiently activated to self-renew in response to bone marrow injury or G-CSF stimulation. After re-establishment of homeostasis, activated HSCs return to dormancy, suggesting that HSCs are not stochastically entering the cell cycle but reversibly switch from dormancy to self-renewal under conditions of hematopoietic stress.
Topics: Adult Stem Cells; Animals; Antigens, Differentiation; Bone Marrow; Bromouracil; Fluorouracil; Green Fluorescent Proteins; Hematopoietic Stem Cells; Homeostasis; Mice; Mice, Transgenic; Uridine
PubMed: 19062086
DOI: 10.1016/j.cell.2008.10.048 -
Photochemistry and Photobiology May 20225-Halouracil, which is a DNA base analog in which the methyl group at the C5 position of thymine is replaced with a halogen atom, has been used in studies of DNA damage.... (Review)
Review
5-Halouracil, which is a DNA base analog in which the methyl group at the C5 position of thymine is replaced with a halogen atom, has been used in studies of DNA damage. In DNA strands, the uracil radical generated from 5-halouracil causes DNA damage via a hydrogen-abstraction reaction. We analyzed the photoreaction of 5-halouracil in various DNA structures and revealed that the reaction is DNA structure-dependent. In this review, we summarize the results of the analysis of the reactivity of 5-halouracil in various DNA local structures. Among the 5-halouracil molecules, 5-bromouracil has been used as a probe in the analysis of photoinduced electron transfer through DNA. The analysis of groove-binder/DNA and protein/DNA complexes using a 5-bromouracil-based electron transfer system is also described.
Topics: Bromouracil; DNA; Thymine; Uracil
PubMed: 34543451
DOI: 10.1111/php.13521 -
International Journal of Molecular... Jul 2019Low-energy electrons (LEEs) of energies ≤30 eV are generated in large quantities by ionizing radiation. These electrons can damage DNA; particularly, they can induce... (Review)
Review
Low-energy electrons (LEEs) of energies ≤30 eV are generated in large quantities by ionizing radiation. These electrons can damage DNA; particularly, they can induce the more detrimental clustered lesions in cells. This type of lesions, which are responsible for a large portion of the genotoxic stress generated by ionizing radiation, is described in the Introduction. The reactions initiated by the collisions of 0.5-30 eV electrons with oligonucleotides, duplex DNA, and DNA bound to chemotherapeutic platinum drugs are explained and reviewed in the subsequent sections. The experimental methods of LEE irradiation and DNA damage analysis are described with an emphasis on the detection of cluster lesions, which are considerably enhanced in DNA-Pt-drug complexes. Based on the energy dependence of damage yields and cross-sections, a mechanism responsible for the clustered lesions can be attributed to the capture of a single electron by the electron affinity of an excited state of a base, leading to the formation of transient anions at 6 and 10 eV. The initial capture is followed by electronic excitation of the base and dissociative attachment-at other DNA sites-of the electron reemitted from the temporary base anion. The mechanism is expected to be universal in the cellular environment and plays an important role in the formation of clustered lesions.
Topics: Antineoplastic Agents; Bromouracil; Carboplatin; Cisplatin; DNA; DNA Breaks, Double-Stranded; Electrons; Eukaryotic Cells; Humans; Oligonucleotides; Oxaliplatin; Plasmids; Radiation, Ionizing; Radiation-Sensitizing Agents
PubMed: 31370253
DOI: 10.3390/ijms20153749 -
Methods (San Diego, Calif.) Feb 2019Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular... (Review)
Review
Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. This review focuses on the methodology used to specifically measure RNA decay at a global level. By comparing and contrasting approaches and describing the experimental protocols in a modular manner, we intend to provide both experienced and new researchers to the field the ability to combine aspects of various protocols to fit the unique needs of biological questions not addressed by current methods.
Topics: Animals; Biotin; Bromouracil; Cell Line; Click Chemistry; High-Throughput Nucleotide Sequencing; Humans; RNA Stability; RNA, Messenger; Staining and Labeling; Thiouracil; Thiouridine; Transcriptome; Uracil; Uridine
PubMed: 30529548
DOI: 10.1016/j.ymeth.2018.12.001 -
Molecules (Basel, Switzerland) Nov 2019Homonucleoside analogues - and - having a (5-methoxycarbonyl)isoxazolidine framework were synthesized via the 1,3-dipolar cycloaddition of nucleobase-derived nitrones...
Homonucleoside analogues - and - having a (5-methoxycarbonyl)isoxazolidine framework were synthesized via the 1,3-dipolar cycloaddition of nucleobase-derived nitrones with methyl acrylate. Hydrogenolysis of the isoxazolidines containing thymine, dihydrouracil, theophylline and adenine moieties efficiently led to the formation of the respective γ-lactam analogues. γ-Lactam analogues having 5-bromouracil and 5-chlorouracil fragments were synthesized by treatment of uracil-containing γ-lactams with NBS and NCS. Isoxazolidine and γ-lactam analogues of homonucleosides obtained herein were evaluated for activity against a broad range of DNA and RNA viruses. None of the compounds that were tested exhibited antiviral or cytotoxic activity at concentrations up to 100 µM. The cytostatic activities of all compounds toward nine cancerous cell lines was tested. γ-Lactams - (Cl-Ura) and - (Theo) appeared the most active toward pancreatic adenocarcinoma cells (Capan-1), showing IC values 21.5 and 18.2 µM, respectively. Isoxazolidine - (Cl-Ura) inhibited the proliferation of colorectal carcinoma (HCT-116).
Topics: Antineoplastic Agents; Isoxazoles; Lactams; Molecular Structure; Nucleosides; Spectrum Analysis
PubMed: 31698778
DOI: 10.3390/molecules24224014 -
Bioorganic & Medicinal Chemistry Jan 20135-Bromouracil ((Br)U) was incorporated into three types of synthetic RNA and the products of the photoirradiated (Br)U-containing RNAs were investigated using HPLC and...
5-Bromouracil ((Br)U) was incorporated into three types of synthetic RNA and the products of the photoirradiated (Br)U-containing RNAs were investigated using HPLC and MS analysis. The photoirradiation of r(GCA(Br)UGC)(2) and r(CGAA(Br)UUGC)/r(GCAAUUCG) in A-form RNA produced the corresponding 2'-keto adenosine ((keto)A) product at the 5'-neighboring nucleotide, such as r(GC(keto)AUGC) and r(CGA(keto)AUUGC), respectively. The photoirradiation of r(CGCG(Br)UGCG)/r(C(m)GCAC(m)GCG) in Z-form RNA produced the 2'-keto guanosine ((keto)G) product r(CGC(keto)GUGCG), whereas almost no products were observed from the photoirradiation of r(CGCG(Br)UGCG)/r(C(m)GCAC(m)GCG) in A-form RNA. The present results indicate clearly that hydrogen (H) abstraction by the photochemically generated uracil-5-yl radical selectively occurs at the C2' position to provide a 2'-keto RNA product.
Topics: Bromouracil; Chromatography, High Pressure Liquid; Light; RNA; Spectrometry, Mass, Electrospray Ionization
PubMed: 23266180
DOI: 10.1016/j.bmc.2012.11.010 -
Chembiochem : a European Journal of... Mar 2022In this study, we investigated the photoreaction of U in a pyrene-labeled DNA duplex, RNA duplex, and DNA/RNA hybrids. We found that the photoreactivity of U changed...
In this study, we investigated the photoreaction of U in a pyrene-labeled DNA duplex, RNA duplex, and DNA/RNA hybrids. We found that the photoreactivity of U changed dramatically from hydrogen abstraction to cross-linking by changing the conformation of the duplex from the B-form to the A-form. Among three A-form structures, the largest amount of cross-linked products was observed when U was incorporated into the RNA strand and the pyrene was conjugated to the 5' end of the DNA. These results indicate that the contact manner of pyrene was different between A- and B-form duplexes. This is a rare example of the use of the reactivity of bromouracil to analyze the contact between a small molecule with a weak binding affinity and a nucleic acid.
Topics: Circular Dichroism; DNA; Nucleic Acid Conformation; Pyrenes; RNA
PubMed: 35080796
DOI: 10.1002/cbic.202100626 -
The Biochemical Journal Aug 19885-Bromo-2'-deoxyuridine triphosphate (Br-dUTP) and dTTP are used interchangeably for DNA synthesis in vitro by the Klenow fragment of Escherichia coli DNA polymerase I....
5-Bromo-2'-deoxyuridine triphosphate (Br-dUTP) and dTTP are used interchangeably for DNA synthesis in vitro by the Klenow fragment of Escherichia coli DNA polymerase I. When DNA containing Br-dUMP instead of dTMP at a few preselected sites is transfected into competent bacteria, no mutation occurs, indicating that in vivo E. coli DNA polymerase always places a dAMP residue in front of any unrepaired Br-dUMP residue. On the other hand, in vitro Br-dUTP can also replace dCTP, but only with difficulty: when dCTP is absent, Br-dUMP can be forced in front of a dGMP residue, but the Klenow polymerase pauses before and after addition of Br-dUMP. Transfection into E. coli of the substituted DNA leads to the expected G----A transitions. These mutations can easily be targeted by using a suitable primer and the correctly chosen mix of deoxynucleoside triphosphates containing Br-dUTP. When Br-dUMP has been placed in front of a dGMP residue, the mutation yield is not 100%, showing a partial repair of the transfected DNA before it is replicated. Advantage can be taken of this partial repair to prepare a set of different mutations within a target region in a single experiment.
Topics: Base Composition; Base Sequence; Bromouracil; DNA; DNA Repair; Deoxyuracil Nucleotides; Growth Hormone; Molecular Sequence Data; Mutation; Templates, Genetic; Transfection
PubMed: 3178734
DOI: 10.1042/bj2530637 -
Scientific Reports Oct 2018We studied photochemical reactions of U-substituted G-quadruplex (G4) DNA substrates with two pyrene-substituted polyazamacrocyclic ligands, M-1PY and M-2PY. Both...
We studied photochemical reactions of U-substituted G-quadruplex (G4) DNA substrates with two pyrene-substituted polyazamacrocyclic ligands, M-1PY and M-2PY. Both ligands bind to and stabilize G4-DNA structures without altering their folding topology, as demonstrated by FRET-melting experiments, fluorimetric titrations and CD spectroscopy. Notably, the bis-pyrene derivative (M-2PY) behaves as a significantly more affine and selective G4 ligand, compared with its mono-pyrene counterpart (M-1PY) and control compounds. Upon short UVA irradiation (365 nm) both ligands, in particular M-2PY, efficiently sensitize photoreactions at U residues incorporated in G4 structures and give rise to two kinds of photoproducts, namely DNA strand cleavage and covalent ligand-DNA photoadducts. Remarkably, the photoinduced strand cleavage is observed exclusively with G4 structures presenting U residues in lateral or diagonal loops, but not with parallel G4-DNA structures presenting only propeller loops. In contrast, the formation of fluorescent photoadducts is observed with all U-substituted G4-DNA substrates, with M-2PY giving significantly higher yields (up to 27%) than M-1PY. Both ligand-sensitized photoreactions are specific to U-modified G4-DNA structures with respect to double-stranded or stem-loop substrates. Thus, ligand-sensitized photoreactions with U-substituted G4-DNA may be exploited (i) as a photochemical probe, allowing "photofootprinting" of G4 folding topologies in vitro and (ii) for covalent trapping of G4 structures as photoadducts with pyrene-substituted ligands.
Topics: Bromouracil; DNA; DNA Adducts; G-Quadruplexes; Humans; Kinetics; Ligands; Models, Molecular; Mutation; Photochemical Processes; Telomere; Uridine
PubMed: 30361545
DOI: 10.1038/s41598-018-34141-z -
Philosophical Transactions. Series A,... Apr 20175-Bromouracil is a nucleobase analogue that can replace thymine in DNA strands and acts as a strong radiosensitizer, with potential applications in molecular biology and...
5-Bromouracil is a nucleobase analogue that can replace thymine in DNA strands and acts as a strong radiosensitizer, with potential applications in molecular biology and cancer therapy. Here, the deactivation of 5-bromouracil after ultraviolet irradiation is investigated in the singlet and triplet manifold by accurate quantum chemistry calculations and non-adiabatic dynamics simulations. It is found that, after irradiation to the bright * state, three main relaxation pathways are, in principle, possible: relaxation back to the ground state, intersystem crossing (ISC) and C-Br photodissociation. Based on accurate MS-CASPT2 optimizations, we propose that ground-state relaxation should be the predominant deactivation pathway in the gas phase. We then employ different electronic structure methods to assess their suitability to carry out excited-state dynamics simulations. MRCIS (multi-reference configuration interaction including single excitations) was used in surface hopping simulations to compute the ultrafast ISC dynamics, which mostly involves the * and * states.This article is part of the themed issue 'Theoretical and computational studies of non-equilibrium and non-statistical dynamics in the gas phase, in the condensed phase and at interfaces'.
Topics: Absorption, Physicochemical; Bromouracil; Electrons; Models, Molecular; Molecular Conformation; Quantum Theory; Ultraviolet Rays
PubMed: 28320905
DOI: 10.1098/rsta.2016.0202